Each neuron is comprised of a nucleus within a body, or soma, a long fiber called the axon, and a varying number of branching fibers called dendrites, which extend out to other neurons. A single neuron can make numerous contacts with other neurons and tissues. For example, every new thought process is handled by a new set of synaptic connections. Memory itself is a set of synaptic connections engraved in the network of neurons.
Dendrites are specialized extensions of the neuronal soma that contain components of the cellular machinery involved in RNA and protein metabolism, as well as a distinctive set of mRNAs. Increasingly, more detailed molecular analyses of dendrites have shown that a subset of cellular RNAs are transported into dendrites where they can be translated into protein at specialized areas following synaptic stimulation (Aakalu et al., 2001, Neuron 30:489-502; Bassell et al., 1998, J. Neurosci. 18:251-65; Crino et al., 1996, Neuron 17:1173-87; Huber et al., 2000, Science 288:1254-7; Job et al., 2001, Proc. Natl. Acad. Sci. U.S.A 98:13037-42; Martin et al., 1997, Cell, 91:927-38). Several of these mRNAs play central roles in synaptic transmission.
In mammalian neurons, the firing of an action potential requires the coordinated gating of at least a dozen different classes of voltage-gated ion channels. The integration of these currents manifests itself as characteristic input-output properties intrinsic to each neuron. One such firing property of some neurons, such as those in the hippocampus (Gu et al., 2007, J Physiol 580(Pt.3):859-82. Epub 2007 Feb. 15; Lancaster et al., 1987, The Journal of Physiology 389: 187-203; Storm, J. F., 1987, The Journal of Physiology 385: 733-759) or cerebellum (Callaway et al., 1997, Journal of Neurophysiology 77: 145-152; Cavelier et al., 2002, The Journal of Physiology 540: 57-72), is the ability to initiate repetitive burst firing in response to depolarizing current. Voltage clamp analysis suggests that the small net inward current that drives the depolarizing momentum is a result of a subtle balance of the sum of inward and outward postspike currents (Swensen et al., 2003, J Neurosci 23: 9650-9663). Although short-term and long-term feedback mechanisms exist to preserve burst firing (Swensen et al., 2005, J Neurosci 25: 3509-3520), it is known that relatively small changes in the size of an individual current may have a dramatic impact on firing activity (Burdakov et al., 2002, J Neurosci 22: 6380-6387).
One transient current activated during the falling phase of the action potential is the BKCa channel. In the central nervous system, BKCa channels are localized to the cell soma as well as the pre- and post-synaptic terminals of neurons where they regulate fundamental neuronal functions such as burst firing, neurotransmitter release, shaping action potential waveforms, and frequency tuning (Salkoff et al., 2006, Nature Reviews 7: 921-931). Native channels are assembled as tetramers of the pore-forming α-subunits encoded by a single gene, KCNMA1 (previously called slo-1), (Atkinson et al., 1991, Science 253: 551-555; Butler et al., 2003, Science 261: 221-224). This gene is subject to vast tissue—(Tseng-Crank et al., 1994, Neuron 13: 1315-1330) and cell-specific alternative splicing (Navaratnam et al., 1997, Neuron 19: 1077-1085; Rosenblatt et al., 1997, Neuron 19: 1061-1075). The resulting functional heterogeneity in BKCa channel currents is due in part to these splicing events generating BKCa channels with altered Ca2+-sensitivity and gating kinetics for (review see Salkoff et al., 2006, Nature Reviews 7: 921-931; Shipston, M. J., 2001, Trends Cell Biol 11: 353-358), as well as altered channel trafficking (Kwon et al., 2004, Proceedings of the National Academy of Sciences of the United States of America 101: 15237-15242; Zarei et al., 2004, Proceedings of the National Academy of Sciences of the United States of America 101: 10072-10077). Channel differences also arise from modulation via a family of tissue-specific auxiliary β-subunits (Fettiplace et al., 1999, Annu Rev Physiol 61: 809-834).
Currently, the functional diversity among BKCa channels in neurons is not fully resolved, but the expression and subcellular distribution patterns of splice variants are expected to be one functionally significant factor in determining their characteristic input-output properties. Indeed, some K+ channels, for example BKCa (Poolos et al., 1999, Neurosci 19: 5205-5212) or A-type (Colbert et al., 1999, J Neurosci 19: 8163-8171; Hoffman et al., 1998, J Neurosci 18: 3521-3528), establish functional gradients within the dendritic processes of mature neurons where they transform the shape of local synaptic potentials or size of somatic action potentials (Gulledge et al., 2005, Journal of Neurobiology 64: 75-90; Johnston et al., 2000, Epilepsia 41: 1072-1073).
A mutation in the gene encoding the pore-forming subunit of BKCa channels has been linked to a variety of human diseases, including autism (Laumonnier et al., 2006, Am J Psychiatry 163:1622-1629), coexistent generalized epilepsy and paroxysmal dyskinesia (Du et al., 2005, Nat Genet. 37: 733-738). In genetic studies, mice lacking the KCNMA1 gene display several dysfunctions, such as progressive hearing loss, cerebellar ataxia, elevated blood pressure and reduced burst firing in cerebellar Purkinje neurons (Sausbier et al., 2005, Circulation 112:60-68; Sausbier et al., 2004, PNAS 101: 9474-9478; Ruttiger et al., 2004, PNAS 101:12922-12927). Changes in BK channel expression levels have also been associated with multiple clinical disorders in humans. The expression levels of BK channels are elevated in human glioma biopsies; the increase expression of the BK channels correlates directly to malignancy grade of the tumor (Liu et al., 2002, J Neurosci 22:1840-1849; Weaver et al., 2004, J Neurosci Res 78:224-234). In ageing human coronary smooth muscle, the expression levels of BK channels are greatly reduced (Marie et al., 2001). The decrease in BK channels expression is believed to increase the risk of coronary spasm and myocardial ischemia in older people.
Despite the existing knowledge in the art about the significant clinical and functional roles associated with BK channels and other neuronal proteins, the functional significance of mRNA isoforms encoding these neuronal proteins has not been addressed. Thus, there is a need in the art for a better understanding of the function and role of mRNA isoforms in order to facilitate the study, diagnosis and therapeutic treatment of pathologies of neurons. This invention addresses that need.